CN104379737A - Variant alpha amylases with enhanced activity on starch polymers - Google Patents

Abstract

Described are variants of alpha-amylase enzymes for use in industrial processes, such as liquefaction of starch. The alpha-amylase variants have increased specific activity allowing the more rapidly reduction of peak viscosity during liquefaction processes. The alpha-amylase is modified by introducing into the amino sequence of a parent Family 13 alpha-amylase polypeptide a mutation at an amino acid residue in the starch-binding groove; wherein the starch-binding groove is formed by amino acid residues in the alpha-helix preceding the first beta-strand in the A domain, the loop between the sixth alpha-helix and the seventh beta-strand in the A domain, the loop between the seventh alpha-helix and the eighth beta-strand in the A domain, and the loop connecting the A domain and the C domain; and wherein the mutation alters the binding of starch to the variant alpha amylase polypeptide compared to the parental alpha amylase polypeptide.

Description

Starch polymer is had to the variant α-amylase of the activity of enhancing

Right of priority

The U.S. Provisional Patent Application No.61/657 that patent application claims was submitted on June 8th, 2012, the right of priority of 501, this patent application is incorporated in full accordingly by reference.

Technical field

The invention describes composition and the method for the variant alpha amylase of the commercial run related to for such as starch liquefacation.Described alpha-amylase variants has the specific activity of enhancing, thus allows to reduce peak viscosity quickly in liquefaction process.

Background technology

α-amylase is used for multiple industry and business process, comprises starch liquefacation, yarn fabric destarch, food mfg, clothes cleaning and dish washing.In this type of application, α-amylase decomposable asymmetric choice net starch and discharge less carbohydrate.But starch bundle can resistance to α-amylasehydrolysis, because enzyme forecloses by organized starch polymer.Therefore, use the α-amylase with the specific activity of enhancing slightly can only improve Starch Hydrolysis, because it and the problem of unresolved accessibility.Need the α-amylase of more effectively touching the intrafascicular starch polymer of starch.

Summary of the invention

The present composition and method relate to variant alpha amylase polypeptide and using method thereof.The many aspects of the compositions and methods of the invention and embodiment are summarised in the paragraph of following independent numbering, and comprise its using method.

1. in one aspect, provide a kind of method producing variant alpha amylase polypeptide, comprising: the amino-acid residue place of aminoacid sequence in starch engagement groove to parent family 13 α-amylase polypeptide introduces sudden change; Wherein starch engagement groove is by the 7th ring between alpha-helix and Article 8 beta chain in the 6th ring between alpha-helix and Article 7 beta chain, A structural domain in the amino-acid residue in the alpha-helix in A structural domain before Article 1 beta chain, A structural domain, and the ring connecting A structural domain and C-structure territory is formed; And wherein described sudden change changes the combination of starch and variant alpha amylase polypeptide compared with parent alpha-amylase polypeptide.

2. in some embodiments of the method for the 1st section, starch engagement groove correspond to amino-acid residue 1-6,36,38,91-97,224-226,249-257,278-282,309-320,354-359,391 and 395-402, relevant numbering is see SEQ ID NO:2; Amino-acid residue 1-6,37,39,92-98,227-229,252-260,281-285,312-323,357-362,391 and 395-402, relevant numbering is see SEQ ID NO:3; Or amino-acid residue 1-4,36,38,91-97,226-228,251-259,280-284,311-322,356-361,363,391 and 395-402, relevant numbering is see SEQ ID NO:4.

3. in some embodiments of the method for aforementioned paragraphs arbitrary section, suddenly change in the amino-acid residue corresponding to amino-acid residue 92,251,254,256,317,318,320 or 321, relevant numbering is see SEQ ID NO:4.

4., in some embodiments of the method for aforementioned paragraphs arbitrary section, sudden change is corresponding to the position of R251 or K256 by the different radical amino acid replacement of wildtype residues, and relevant numbering is see SEQ ID NO:4.

5. in some embodiments of the method for aforementioned paragraphs 1-3 arbitrary section, sudden change is replaced by wildtype residues L in the position corresponding to the position 92 in SEQ ID NO:4, corresponding to the position of the position 251 in SEQ ID NO:4 by wildtype residues A, C, D, E, F, G, H, L, M, N, Q, S, T or W replaces, and is corresponding to the position of the position 254 in SEQ ID NO:4 by wildtype residues H, K, W or Y replaces, and is corresponding to the position of the position 256 in SEQ ID NO:4 by wildtype residues A, E, T or V replaces, and is replaced by wildtype residues C or R in the position corresponding to the position 317 in SEQ ID NO:4, is corresponding to the position of the position 318 in SEQ ID NO:4 by wildtype residues A, F, H, K, Q or R replaces, and is corresponding to the position of the position 320 in SEQ ID NO:4 by wildtype residues A, H, M, N or P replaces, or is corresponding to the position of the position 321 in SEQ ID NO:4 by wildtype residues D, G, I or T replaces.

6. in some embodiments of the method for aforementioned paragraphs 1-3 arbitrary section, sudden change corresponds to S92L, R251A, R251C, R251D, R251E, R251F, R251G, R251H, R251L, R251M, R251N, R251Q, R251S, R251T, or R251W, T254H, T254K, T254W, or T254Y, K256A, K256E, K256T, or K256V, S317C or S317R, N318A N318F, N318H, N318K, N318Q, or N318R, T320A, T320H, T320M, T320N, or T320P, and/or K321D, K321G, K321I, or K321T, relevant numbering is see SEQ ID NO:4.

7. in some embodiments of the method for aforementioned paragraphs arbitrary section, variant is also included in the wt amino acid residue of the one or more positions corresponding to N88, A252, A253, N308, A316, S357, T400, R402 and D403, and relevant numbering is see SEQ IDNO:4.

8. in some embodiments of the method for aforementioned paragraphs 1-7 arbitrary section, variant is also included in the wt amino acid residue of the one or more positions corresponding to N4, G5, T38, N93, G94, I95, Q96, V97, Y230, G255, V315, P319, A322, L354, T355, R356, G359, Y396, A397, Y398, G399 and the Q401 in SEQ ID NO:4, and relevant numbering is see SEQ ID NO:4.

9. in some embodiments of the method for aforementioned paragraphs 1-8 arbitrary section, variant is also included in N, the A in position 252, the A in position 253, the N in position 308, the A in position 316, the S in position 357, the T in position 400, the R in the position 402 or D in position 403 of position 88, and relevant numbering is see SEQ ID NO:4.

10. in some embodiments of the method for aforementioned paragraphs 1-9 arbitrary section, variant is also included in the N of position 4, at the G of position 5, at the T of position 38, at the N of position 93, at the G of position 94, at the I of position 95, at the Q of position 96, at the V of position 97, at the Y of position 230, at the G of position 255, at the V of position 315, at the P of position 319, at the A of position 322, at the L of position 354, at the T of position 355, at the R of position 356, at the G of position 359, at the Y of position 396, at the A of position 397, at the Y of position 398, at the G of position 399, with the Q in position 401, relevant numbering is see SEQ ID NO:4.

11. in some embodiments of the method for aforementioned paragraphs arbitrary section, use cyclodextrin to measure relative starch and combine.

12. in some embodiments of the method for aforementioned paragraphs arbitrary section, variant have with SEQ IDNO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

13. in some embodiments of the method any one of aforementioned claim, parent have with SEQ ID NO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

14. in some embodiments of the method for aforementioned paragraphs arbitrary section, and variant shows the starch liquefacation of improvement, mashing or clean-up performance compared with parent.

15. in some embodiments of the method for aforementioned paragraphs arbitrary section, and variant shows the hydrolytic activity strengthened amylopectin substrate compared with parent.

16. on the other hand, provides the variant alpha amylase polypeptide produced by the method described in aforementioned claim.

17. on the other hand, provides the variant of parent family 13 α-amylase polypeptide, and it comprises sudden change in starch engagement groove; Wherein starch engagement groove is by the 7th ring between alpha-helix and Article 8 beta chain in the 6th ring between alpha-helix and Article 7 beta chain, A structural domain in the amino-acid residue in the alpha-helix in A structural domain before Article 1 beta chain, A structural domain, and the ring connecting A structural domain and C-structure territory is formed; Wherein described sudden change changes the combination of starch and variant alpha amylase polypeptide compared with parent alpha-amylase polypeptide.

18. in some embodiments of the variant alpha amylase polypeptide of the 17th section, starch engagement groove correspond to amino-acid residue 1-6,36,38,91-97,224-226,249-257,278-282,309-320,354-359,391 and 395-402, relevant numbering is see SEQID NO:2; Amino-acid residue 1-6,37,39,92-98,227-229,252-260,281-285,312-323,357-362,391 and 395-402, relevant numbering is see SEQ ID NO:3; Or amino-acid residue 1-4,36,38,91-97,226-228,251-259,280-284,311-322,356-361,363,391 and 395-402, relevant numbering is see SEQ ID NO:4.

19. in some embodiments of the variant alpha amylase polypeptide of aforementioned claim 17 or 18, suddenly change in the amino-acid residue corresponding to amino-acid residue 92,251,254,256,317,318,320 or 321, and relevant numbering is see SEQ ID NO:4.

20. in some embodiments of the variant alpha amylase polypeptide any one of aforementioned claim 17-19, and sudden change is corresponding to the position of R251 or K256 by the different radical amino acid replacement of wildtype residues, and relevant numbering is see SEQ ID NO:4.

21. in some embodiments of the variant alpha amylase polypeptide of aforementioned paragraphs 17-19 arbitrary section, sudden change is replaced by wildtype residues L in the position corresponding to the position 92 in SEQ ID NO:4, corresponding to the position of the position 251 in SEQ ID NO:4 by wildtype residues A, C, D, E, F, G, H, L, M, N, Q, S, T or W replaces, and is corresponding to the position of the position 254 in SEQ ID NO:4 by wildtype residues H, K, W or Y replaces, and is corresponding to the position of the position 256 in SEQ ID NO:4 by wildtype residues A, E, T or V replaces, and is replaced by wildtype residues C or R in the position corresponding to the position 317 in SEQ ID NO:4, is corresponding to the position of the position 318 in SEQID NO:4 by wildtype residues A, F, H, K, Q or R replaces, and is corresponding to the position of the position 320 in SEQ ID NO:4 by wildtype residues A, H, M, N or P replaces, or is corresponding to the position of the position 321 in SEQ ID NO:4 by wildtype residues D, G, I or T replaces.

22. in some embodiments of the variant alpha amylase polypeptide of aforementioned paragraphs 17-19 arbitrary section, sudden change corresponds to S92L, R251A, R251C, R251D, R251E, R251F, R251G, R251H, R251L, R251M, R251N, R251Q, R251S, R251T, or R251W, T254H, T254K, T254W, or T254Y, K256A, K256E, K256T, or K256V, S317C or S317R, N318AN318F, N318H, N318K, N318Q, or N318R, T320A, T320H, T320M, T320N, or T320P, or K321D, K321G, K321I, or K321T, relevant numbering is see SEQ ID NO:4.

23. in some embodiments of the variant alpha amylase polypeptide of aforementioned paragraphs 17-22 arbitrary section, variant is also included in the wt amino acid residue of the one or more positions corresponding to N88, A252, A253, N308, A316, S357, T400, R402 and/or D403, and relevant numbering is see SEQ ID NO:4.

24. in some embodiments of the variant alpha amylase polypeptide of aforementioned paragraphs 17-23 arbitrary section, variant is also included in the wt amino acid residue of the one or more positions corresponding to N4, G5, T38, N93, G94, I95, Q96, V97, Y230, G255, V315, P319, A322, L354, T355, R356, G359, Y396, A397, Y398, G399 and/or the Q401 in SEQ ID NO:4, and relevant numbering is see SEQ ID NO:4.

25. in some embodiments of the variant alpha amylase polypeptide of aforementioned paragraphs 17-24 arbitrary section, variant is also included in N, the A in position 252, the A in position 253, the N in position 308, the A in position 316, the S in position 357, the T in position 400, the R in the position 402 and/or D in position 403 of position 88, and relevant numbering is see SEQ ID NO:4.

26. in some embodiments of the variant alpha amylase polypeptide of aforementioned paragraphs 17-25 arbitrary section, variant is also included in the N of position 4, at the G of position 5, at the T of position 38, at the N of position 93, at the G of position 94, at the I of position 95, at the Q of position 96, at the V of position 97, at the Y of position 230, at the G of position 255, at the V of position 315, at the P of position 319, at the A of position 322, at the L of position 354, at the T of position 355, at the R of position 356, at the G of position 359, at the Y of position 396, at the A of position 397, at the Y of position 398, at the G of position 399, and/or the Q in position 401, relevant numbering is see SEQ ID NO:4.

27. in some embodiments of the variant of aforementioned paragraphs 17-26 arbitrary section, variant have with SEQ ID NO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

28. in some embodiments of the variant of aforementioned paragraphs 17-27 arbitrary section, parent have with SEQ ID NO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

29. in some embodiments of the variant of aforementioned paragraphs 17-28 arbitrary section, and variant shows the starch liquefacation of improvement, mashing or clean-up performance compared with parent.

30. in some embodiments of the variant of aforementioned paragraphs 17-29 arbitrary section, and variant shows the hydrolytic activity strengthened amylopectin substrate compared with parent.

31. in yet another aspect, provides a kind ofly to comprise the variant alpha amylase polypeptide of aforementioned paragraphs 17-30 arbitrary section and the composition of at least one preparaton.

By description of the invention and accompanying drawing, the these and other aspects of the compositions and methods of the invention and embodiment will be apparent.

Accompanying drawing explanation

Fig. 1 is the engineered diastatic belt pattern derived from walsh fireball bacterium (Pyrococcus woesei), shows the position of the cyclodextrin of combination.

Fig. 2 shows the position of aminoacid sequence in engineered walsh fireball bacterium (P.woesei) amylase and second structure characteristic.

Fig. 3 is the comparison of some families 13 α-amylase [Geobacillus stearothermophilus (Geobacillus stearothermophilus) (being called bacstearothermophilus (Bacillus stearothermophilus) in the past) α-amylase, Bacillus licheniformis (Bacilluslicheniformis) α-amylase (LAT) α-amylase and Cytophaga species (Cytophaga sp.) α-amylase (AAF00561.1, GI#6006681)] using Clustal W to carry out with default parameters.

Fig. 4 is the club shaped structure of bacstearothermophilus (B.stearothermophilis) amylase (AmyS), shows the groove residue be positioned on the left of molecule highlighted with overstriking club.Binding Capacity district (that is, avtive spot crack) is positioned on the right side of molecule.

Fig. 5 is the club shaped structure of bacstearothermophilus amylase (AmyS) when observing groove downwards, and the groove wherein forming residue highlights with overstriking club.

Fig. 6 is histogram, shows the PI distribution of the activity value of all SEL variants of expressing PI>0.3.

Fig. 7 is histogram, shows and in slot is put, only has displacement and the PI distribution expressing the activity value of the SEL variant of PI>0.3.

Fig. 8 A and 8B is form, shows the result using amylopectin substrate to screen the independent variant obtained from SEL library.

Fig. 9 A and 9B is form, shows the result using pullulan substrate to screen the independent variant obtained from SEL library.

the brief description of sequence

SEQ ID NO:1 shows the aminoacid sequence of the diastatic mature form of walsh fireball bacterium.

SEQ ID NO:2 shows the aminoacid sequence of the mature form of bacillus licheniformis alpha-amylase (LAT) α-amylase.

SEQ ID NO:3 shows the aminoacid sequence of the mature form of Geobacillus stearothermophilus (Geobacillusstearothermophilus) (being called bacstearothermophilus (Bacillusstearothermophilus) in the past) α-amylase.

SEQ ID NO:4 shows the aminoacid sequence of the mature form of Cytophaga species α-amylase (AAF00561.1, GI#6006681).

SEQ ID NO:5 shows the aminoacid sequence of the mature form of variant walsh fireball bacterium amylase uPWA.

Embodiment

1. introduction

The compositions and methods of the invention relate to variant alpha amylase polypeptide, and it comprises sudden change being arranged in the newfound starch engagement groove on the molecular locus relative with substrate binding site.Sudden change in described starch engagement groove changes the combination of variant alpha amylase polypeptide and starch bundle, thus changes the performance of molecule in such as activity, thermostability, pH stability, washing composition stability, Ca-dependent etc.

These and other aspect of described composition and method is hereafter being described in detail.

2. definition and abbreviation

Describe in detail according to this, apply abbreviation below and definition.Note, unless the context clearly indicates otherwise, otherwise singulative " ", " one " and " described " comprise and multiplely refer to thing.Therefore, such as, mention that " enzyme " comprises multiple this kind of enzyme, and mention " dosage ", comprise and mention one or more dosage and its equivalent well known by persons skilled in the art, etc.

This document is organized into multiple chapters and sections, so that read; But reader will recognize that, the statement carried out in chapters and sections can be applied to other chapters and sections.Therefore, the title that the different chapters and sections of the disclosure use should not be construed as restrictive.

Unless otherwise defined, otherwise all technology used herein and scientific terminology have usual the understood implication of those of ordinary skill in the art.For the sake of clarity, the following abbreviation of definition and/or term:

2.1 abbreviations/acronym

Following abbreviation/acronym has following implication, unless specified otherwise herein:

CDNA complementary DNA

DNA thymus nucleic acid

EC EC

EDTA ethylenediamine tetraacetic acid (EDTA)

GA glucoamylase

IPTG isopropyl ss-D-thiogalactoside

KDa kilodalton

LAT B. licheniformis amylase

MW molecular weight

The diastatic index of MWU improvement; 1.6 × 10 ^-5mg/ MWU=activity unit

NOBS nonanoyloxybenzenesulfonate

NTA nitriloacetic acids

PEG polyoxyethylene glycol

PI iso-electric point

PVA gathers (vinyl alcohol)

PVP PVP

RNA Yeast Nucleic Acid

SAS alkylsulfonate

SDS-PAGE SDS-PAGE

Sp. species

W/v weight/volume

W/w w/w

V/v volume/volume

Wt% % by weight

DEG C degree Celsius

H ₂o water

DH ₂o or DI deionized water

DIH ₂the deionized water that O Milli-Q filters

G or gm gram

μ g microgram

Mg milligram

Kg kilogram

μ L and μ l microlitre

ML and ml milliliter

Mm millimeter

dust

μm micron

M mole

MM mmole

μM micromole

U unit

Sec second

Min minute

Hr hour

Ncm newton centimetre

EtOH ethanol

Eq. equivalent

N equivalent concentration

Ds or DS dry solid content

UPWA is derived from the variant alpha amylase of walsh fireball bacterium

PWA is from the α-amylase of walsh fireball bacterium

MWCO molecular weight cutoff

Synchrotron radiation light source, SSRL Stamford (Stanford Synchrotron Radiation Lightsource)

PDB Protein Data Bank

CAZy carbohydrate activity enzyme database

Tris-HCl tri-(methylol) aminomethane hydrochloride

HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

2.2 definition

Term " amylase " or " amylolytic enzyme " refer among other aspects can also the enzyme of degraded of catalytic starch.α-amylase is the lytic enzyme of α-D-(1 → 4) the O-glycosides key in cracking starch.In general, α-amylase (EC 3.2.1.1; α-D-(1 → 4)-dextran glucan hydrolase) be defined as α-D-(1 → 4) the O-glycosides key in cracking starch molecule in a random basis thus generate the inscribe effect enzyme of the polysaccharide of the D-Glucose unit containing three or more (1-4)-α-connections.On the contrary, circumscribed effect amylolytic enzyme is as beta-amylase (EC 3.2.1.2; α-D-(1 → 4)-dextran malto-hydrolase) and some product specificities amylase if maltogenic alpha-amylase (EC 3.2.1.133) is from the non-reducing end cracking polysaccharide molecule of substrate.Beta-amylase, alpha-glucosidase (EC 3.2.1.20; α-D-glucoside glucose hydrolysis enzyme), glucoamylase (EC 3.2.1.3; α-D-(1 → 4)-dextran glucan hydrolase) and product specificities amylase such as maltotetrose glycosides enzyme (EC 3.2.1.60) and MALTOHAXAOASE glycosides enzyme (EC3.2.1.98) malto-oligosaccharide of length-specific can be produced.Some bacterialα-amylases are mainly from starch and relevant α-1,4-dextran produces maltotetrose (G4), maltopentaose (G5) or MALTOHAXAOASE (G6), and they are converted into glucose and/or maltose as final product by most of α-amylase further.For the object of the compositions and methods of the invention, in real inscribe effect α-amylase and do not distinguish between G4, G5, G6 and similar amylase.

Statement " family 13 sample amylase " refers to have classifying as CAZy family 13 amylase and/or being called any amylase of " Termamyl sample " up to now of at least 60% amino acid sequence identity with SEQ ID NO:1-5.Statement " family 13 amylase " refers to and classifies as the diastatic any amylase of CAZy family 13.

Statement " having Bacillus spec (Bacillus sp.) diastatic folding amylase " refers to any amylase with the structure substantially overlapped on SEQ ID NO:2 or 3.

Statement " Termamyl sample " is used in multiple patent application of having announced and patent.

As used herein, term " starch " refers to any material be made up of the complicated polysaccharide carbohydrate of plant, by having formula (C ₆h ₁₀o ₅) _xthe amylose starch of (wherein X can be any numeral) and amylopectin are formed.Described term comprises plant based material, as cereal, grass, stem tuber and root, and it is more particularly the material obtained from wheat, barley, corn, naked barley, paddy rice, jowar, chaff, cassava (cassava), millet, potato, sweet potato and cassava (tapioca).

Term " starch bundle " refers to a kind of form of starch, and wherein then each starch polymer is such as brought back to life by gelatinization and arrange, and makes the enzymically hydrolyse of their resistance to α-amylase.In some cases, starch intrafascicular polymkeric substance is parallel (that is, alignment), thus enzyme cannot be touched.

Term " amylose starch " refers to the non-branching starch substrates be made up of the glucosyl residue in α-Isosorbide-5-Nitrae bonding.

Term " amylopectin " refers to the branched starch substrate be made up of α-1,6 bonding and α-Isosorbide-5-Nitrae bonding.

About the term " wild-type " of polypeptide, " parent " or " reference " refer to do not comprise at one or more amino acid position place artificial manufacture displacement, insertion or disappearance naturally occurring polypeptide.Similarly, refer to about the term " wild-type " of polynucleotide, " parent " or " reference " the naturally occurring polynucleotide not comprising the artificial nucleosides manufactured and change.But, it should be noted that the polynucleotide of encoding wild type polypeptide, parental polypeptide or reference polypeptide are not limited to naturally occurring polynucleotide, and contain any polynucleotide of encoding wild type polypeptide, parental polypeptide or reference polypeptide.Displacement, insertion or disappearance produce by " reorganization ".

Statement " one or several " means to be less than 10.

Term " variant " about polypeptide refers to the polypeptide being different from specific wild type peptide, parental polypeptide or reference polypeptide because its comprise at one or more amino acid position place artificial manufacture displacement, insertion or disappearance.Similarly, the term " variant " about polynucleotide refers to that nucleotide sequence is different from wild-type polynucleotide, parent polynucleotide or the polynucleotide with reference to polynucleotide of specifying.The identity of wild-type, parent or reference polypeptide or polynucleotide will be apparent from context.Variant is prepared by " reorganization ".

When using about subject cell, nucleic acid, protein or carrier, term " restructuring " shows object by introducing heterologous nucleic acids or protein or changing natural nucleic acid or protein and modified, or the cell-derived cell modified through this type of of controlling oneself.Therefore, such as, reconstitution cell expresses the gene do not existed in the cell of natural (non-recombinant) form, or expresses natural gene with the level or condition that are different from occurring in nature existence.

Term " recovery ", " separation " and " separating " refer to remove from other materials of relevant at least one natural to it as naturally occurring or component compound, protein (polypeptide), cell, nucleic acid, amino acid or other material of specifying or component.

As used herein, term " purifying " refer to be in relatively pure state material (as, isolated polypeptide or polynucleotide), described relatively pure state as purity at least about 90%, purity at least about 95%, purity at least about 98% or purity even at least about 99%.

Under the existence of well-oxygenated environment, sequestrant, under the existence of washing composition, be exposed to high temperature and/or be exposed in the context of pH extreme value, term " stability of enhancing " or " stability of raising " refer to another kind of (namely, reference) amylase compares, and object amylase passes the higher amylolytic activity of maintenance in time.

The ability that enzyme keeps active is after exposure to elevated temperatures referred to about the term " heat-staple " of enzyme and " thermostability ".The thermostability of enzyme (as amylase) is by its transformation period (t _1/2) measure, described transformation period by minute, hour or day in units of provide, enzymic activity loses half under the condition limited during this period.Transformation period by measure be exposed to (that is, standing) high temperature after remaining amylase activity calculate.

" pH scope " about enzyme refers to that enzyme demonstrates the pH value range of catalytic activity under it.

As used herein, relate within predetermined time section (e.g., 15 minutes, 30 minutes, 1 hour) about the term " pH is stable " of enzyme and " pH stability ", enzyme keeps active ability in wide in range pH value range.

As used herein, term " aminoacid sequence " and term " polypeptide ", " protein " and " peptide " are synonyms, and are used interchangeably.When this type of aminoacid sequence shows activity, they can be called as " enzyme ".Use single-letter or the three-letter codes of conventional amino-acid residue, wherein aminoacid sequence provides to carboxyl terminal orientation (that is, N → C) with the aminoterminal of standard.

DNA, RNA, heteroduplex and can the synthetic molecules of coded polypeptide contained in term " nucleic acid ".Nucleic acid can be strand or double-strand, and can be chemical modification object.Term " nucleic acid " and " polynucleotide " are used interchangeably.Because genetic code has degeneracy, therefore a more than codon can be used to carry out encoding particular amino acid, and the present composition and method contain the nucleotide sequence of encoding particular amino acid sequence.Unless otherwise noted, nucleotide sequence provides with 5' to 3' orientation.

Term " homologue " refers to the entity with object aminoacid sequence and object nucleotide sequence with the identity of given extent.Homologous sequence is believed to comprise and object sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identical aminoacid sequence, sequence alignment uses Clustal W (Thompson J.D.et al. (1994) the Nucleic Acids Res.22:4673-4680 (people such as Thompson J.D., 1994, " nucleic acids research ", 22nd volume, 4673-4680 page)) carry out with following default parameters, that is:

Term " hybridization " refers to the chain that nucleic acid base is right and the process that complementary strand occurs during blot hybridization technique and round pcr.Strict hybridization conditions is as follows: 65 DEG C and 0.1X SSC (wherein 1X SSC=0.15M NaCl, 0.015M trisodium citrate, pH 7.0).

Term " saccharification " refers to that Starch Conversion is glucose by enzyme.

Term " liquefaction " refers to the stage in Starch Conversion, and wherein gelation starch is hydrolyzed and produces low-molecular-weight soluble dextrins.Term " polymerization degree " (DP) refers to the number (n) of anhydrous Glucopyranose in given carbohydrate.The example of DP1 is that monose is as glucose and fructose.The example of DP2 is disaccharides, as maltose and sucrose.For object herein, " comparable liquefaction process " refers to the liquefaction process preferably carried out under the conditions such as standardized temperature, pH, concentration of substrate, calcium ion concn under comparable condition.Preferably, comparable liquefaction process compares based on equal protein or equal activity unit, but, in certain embodiments, in comparable liquefaction process protein or activity unit or both all can change.Technician will understand the basis that liquefaction process can be " comparable " institute foundation.

In liquefaction process process, the viscosity of farinaceous size is commonly used for the tolerance that starch transforms to less DP unit.Sometimes statement " initial viscosity " is used herein.Technician is a buzzword by understanding this, and is not intended to literally represent initial viscosity, and refers to the peak viscosity occurred near such as substrate gelatinization point.Initial viscosity is used for distinguishing " final viscosity ", and " final viscosity " is substrate (such as farinaceous size) viscosity at the end of liquefaction process.Therefore, the figure line changed with the time course of liquefaction process by viscosity be it is evident that, initial viscosity or peak viscosity non-immediate occur, but occur afterwards in certain period (be such as less than liquefaction total time 50%), more preferably appearance in 1/3 or even 1/4 or 1/5 before the pact of liquefaction total time.Such as, in liquefaction exemplified here, peak viscosity occurred usually in front ten minutes, added enzyme after at this moment, and viscosity starts to decline.When starch granules is opened in gelatinization technological process, granule interior becomes the impact that is more subject to amylase activity and produces more cracking, thus causes viscosity degradation.

Term " dry solid content " (ds) refers to the total amount by solid substance in dry weight basis slurries.The weight that dry solid content and dry weight basis are typically expressed as measured material accounts for the per-cent of total dry substance weight.Term " slurries " refers to and to be generally in water or similar solvent containing the mixture of insoluble solid at liquid.Usually starch or flour are suspended in group water solution and form slurries, for detection amylase or for liquefaction process.

Term " dextrose equivalent " or " DE " are defined as the per-cent that reducing sugar accounts for total carbohydrates part.Term " extent of polymerization " or " DP " refer to the size of the amylase degrades product of starch.DP is higher, and carbohydrate is more complicated.

Term " blend " or " enzyme blend " refer to the composition of the mixture comprising two or more enzymes, provide the catalytic activity of combination thus, and it contains the activity of often kind of enzyme existing in mixture.Enzyme blend do not need in two or more enzymes each there is equal amount, if but need, enzyme blend can be prepared on the basis of equal protein matter or equivalent amount of active.The catalytic activity of combination can be only cumulative or average, can be maybe antagonism or collaborative.Preferred blends used herein provides at least cumulative catalytic activity, more preferably provides collaborative catalytic effect.

For object herein, " comparable liquefaction process " means to use different or that " contrast " amylase carries out similar procedure under controlled and defined terms (such as in temperature, pH, calcium ion concn and concentration of substrate), and it can compared with object liquefaction process.

As used herein, one " α-amylase unit (AAU) " is the amount of the bacterial alpha amylase activity under prescribed conditions needed for per minute hydrolysis 10mg starch.

As used herein, the term " conversions ", " stable conversion " and " transgenosis " that use about cell mean containing to be integrated in its genome or as the cell of non-natural (e.g., the allos) nucleotide sequence through how carrying for the episome remained.

When nucleotide sequence is inserted cell, term " introducing " means " transfection " known in the art, " conversion " or " transduction ".

" host strain " or " host cell " is by expression vector, phage, virus or other DNA construct, comprises the polynucleotide introducing organism wherein of the polypeptide (e.g., amylase) that coding is paid close attention to.Exemplary host strain is bacterial cell.Term " host cell " comprises the protoplastis produced by cell, the protoplastis of such as Bacillus spec.

Term " allos " about polynucleotide or protein refers to and non-natural is present in polynucleotide in host cell or protein.

Term " endogenous " about polynucleotide or protein refers to and is naturally present in polynucleotide in host cell or protein.

As used herein, term " expression " refers to the process generating polypeptide based on nucleotide sequence.Described process comprises transcribes and translates the two.

" selective marker " or " selectable marker " refers to such gene, and it can express the host cell being conducive to selecting to carry this gene in host.The example of selectable marker includes, but is not limited to biocide (as Totomycin, bleomycin or paraxin) and/or gives the gene of host cell metabolism benefit (as nutritional benefits).

" carrier " refers to the polynucleotide sequence that design is used for being introduced by nucleic acid in one or more cell types.Carrier comprises cloning vector, expression vector, shuttle vectors, plasmid, phage particle, box (cassette) etc.

" expression vector " refers to the DNA construct of the DNA sequence dna comprising encoding target polypeptide, and described encoding sequence is effectively connected to the appropriate control sequences that can realize described DNA and express in suitable host.This control sequence can comprise the sequence of the suitable ribosome bind site on operon sequence that the promotor, the optional control that realize transcribing transcribes, coding mRNA, enhanser and control and transcribe the sequence with translation termination.

Term " effectively connection " means specified ingredients and is in a kind of relation (including but not limited to juxtaposition) allowing them to work by way of expectations.Such as, if the expression of encoding sequence is under the control of adjustment sequence, described adjustment sequence is effectively connected with described encoding sequence.

" signal sequence " is the amino acid whose sequence of the N-end portion being connected to protein, and it promotes that protein secreting is to extracellular.The mature form of extracellular protein does not have signal sequence, and it is cut during secretion process.

As used herein, " biological activity " refers to the sequence with appointment biological activity (such as enzymic activity).

As used herein, " comprising the culturing cell material of α-amylase polypeptide " or similar term refer to and comprise α-amylase polypeptide as the cell pyrolysis liquid of component or supernatant liquor (comprising substratum).Cell material preferably comes comfortable for producing the heterologous host grown in the culture of the object of α-amylase polypeptide.

All references cited herein is incorporated to way of reference clearly.

3. the qualification of new starch binding site in α-amylase

See appended " example ", with the three-dimensional structure (Fig. 1 and 2) of engineered variant alpha amylase of resolving power determination hyperthermophilic archaeon strain (hyperthermophilic archaeon) walsh fireball bacterium.Surprisingly, it is found that cyclodextrin molecular is associated with enzyme in crystal (Fig. 1).Cyclodextrin is not a part for crystallization medium, thus shows that cyclodextrin is attached to molecule during expression or purifying, and keeps together with molecule in crystallisation process.Cyclodextrin molecular is not associated with substrate binding site.On the contrary, it is combined in the groove be arranged on the molecule side relative with substrate binding site.

It should be noted that, the position of the some sudden changes (i.e. D358Y, S359G, R360D, R361K and P372S) in variant walsh fireball bacterium α-amylase near the cyclodextrin combined, thus shows that the sudden change in these positions regulates keying action.Estimate the one or more atoms at institute's coupling collar dextrin by regulating this keying action interior adjacent residues comprises Gln7, Lys8, Gly9, Lys241, Pro278, Phe279, Glu307, Gly308, Gln309, Gly338, Gly339, Ser340, Arg355 and Tyr358.The other residue forming starch engagement groove comprises residue Ser4, Glu5, Tyr236, Gln268, Ser275, Arg276, Asp277, Lys280, Tyr306, Glu334, Thr341, Asp342, Ile343, Asn 356, Asp360 and Lys361.

4. there is the variant alpha amylase of sudden change in starch engagement groove

Be not limited to theory, the compositions and methods of the invention are based on following hypothesis: in three-dimensional structure mentioned above, and cyclodextrin simulation α-amylase is advantageously attached to starch helical bundle wherein.The existence of starch binding domains allows amylase to be associated with starch bundle, and even may slide into failure position along starch bundle, in these positions, enzyme hydrolyzable is combined in the starch chain of classical substrate binding site.Slide into the ability being conducive to the region be hydrolyzed the one dimension that diffusion degree of freedom is reduced to along starch molecule from the three-dimensional diffusion solution is spread.This will strengthen the amylatic persistence of molecule, as by by improve Starch Hydrolysis confirm.Starch via groove combines the destruction that even can promote starch bundle, thus strengthens hydrolysis.In addition, via groove starch combine can give stability to α-amylase, this by such as strengthen thermostability, enhancing the confirmation such as pH stability, the washing composition stability of enhancing, the Ca-dependent of reduction.

The diastatic three-dimensional structure of carbohydrate activity enzyme database (CAZy) family 13 is high conservative, form (see such as Henrissat by three structural domains, B. (1991) Biochem.J.280:309-316 (Henrissat, the people such as B, 1991, " journal of biological chemistry ", the 280th volume, 309-316 page); Henrissat, B.and Bairoch, A. (1996) Biochem.J.316:695-696 (Henrissat, B. and Bairoch, A., " journal of biological chemistry ", the 316th volume, 695-696 page in 1996); Henrissat, B.and Davies, G. (1997) Curr.Opin.Biotech.7:637-644 (Henrissat, B. and Davies, G., " biotechnology neodoxy ", the 7th volume, 637-644 page in 1997); Nagano, N.et al. (2001) Protein Eng.14:845-855 (people such as Nagano, N., calendar year 2001, " protein engineering ", the 14th volume, 845-855 page); Pujadas, G.and Palau, J. (2001) Mol.Biol.Evol 18:38-54 (Pujadas, G. and Palau, J., calendar year 2001, " molecular biology and evolution ", the 18th volume, 38-54 page); WO 94/02597).It is the TIM barrel-like structure held at polypeptide N see Fig. 2, A structural domain.B structural domain is the Article 3 beta chain (β in A structural domain _a3) and the 3rd alpha-helix (α _a3) between great Huan district.C-structure territory comprises the C end of polypeptide.Amylase from Bacillus licheniformis (AmyL), bacstearothermophilus (AmyS) and Cytophaga species is all members of this family.These diastatic comparisons are shown in Figure 3.The example of these diastatic structures is respectively entry 1hxv in Protein Data Bank (Protein Data Bank) and 1bli, there is illustrated the even more obviously cavity feature of the respective regions be arranged on these molecules.

These enzymes each in, there are about 44 the homology residues forming starch engagement groove.7th ring between alpha-helix with Article 8 beta chain and being connected in the ring in A structural domain and C-structure territory in 6th ring between alpha-helix with Article 7 beta chain, A structural domain in the short alpha-helix of these residues in A structural domain before Article 1 beta chain, A structural domain.In variant walsh fireball bacterium amylase, these residues correspond to residue Gln7, Lys8, Gly9, Lys241, Pro278, Phe279, Glu307, Gly308, Gln309, Gly338, Gly339, Ser340, Arg355 and Tyr358.The other residue forming starch engagement groove comprises residue Ser4, Glu5, Tyr236, Gln268, Ser275, Arg276, Asp277, Lys280, Tyr306, Glu334, Thr341, Asp342, Ile343, Asn 356, Asp360 and Lys361 (relevant numbering is see SEQ ID NO:5).In Bacillus licheniformis (B.licheniformis) amylase, these residues correspond to residue 1-6,36,38,91-97,224-226,249-257,278-282,309-320,354-359,391 and 395-402 (relevant numbering see SEQ ID NO:2).In bacstearothermophilus amylase, these residues correspond to residue 1-6,37,39,92-98,227-229,252-260,281-285,312-323,357-362,391 and 395-402 (relevant numbering see SEQ ID NO:3).In Cytophaga species amylase, these residues correspond to residue 1-4,36,38,91-97,226-228,251-259,280-284,311-322,356-361,363,391 and 395-402 (relevant numbering see SEQ ID NO:4).

In certain embodiments, the position that variant of the present invention is corresponding to the position 92,251,254,256,317,318,320 and 321 in SEQ ID NO:4 has one or more displacement.In certain embodiments, described variant is included in position corresponding to R251 and/or K256 in SEQ ID NO:4 by radical amino acid replacement lower for wildtype residues positive polarity.In certain embodiments, wildtype residues L replaces by the position that variant of the present invention is included in corresponding to the position 92 in SEQ ID NO:4, corresponding to the position of the position 251 in SEQ ID NO:4 by wildtype residues A, C, D, E, F, G, H, L, M, N, Q, S, T or W replaces, corresponding to the position of the position 254 in SEQID NO:4 by wildtype residues H, K, W or Y replaces, corresponding to the position of the position 256 in SEQ ID NO:4 by wildtype residues A, E, T or V replaces, in the position corresponding to the position 317 in SEQ ID NO:4, wildtype residues C or R is replaced, corresponding to the position of the position 318 in SEQ ID NO:4 by wildtype residues A, F, H, K, Q or R replaces, corresponding to the position of the position 320 in SEQ ID NO:4 by wildtype residues A, H, M, N, or P displacement, and/or corresponding to the position of the position 321 in SEQ ID NO:4 by wildtype residues D, G, I or T replaces.

In certain embodiments, variant of the present invention comprises corresponding to S92L, R251A, R251C, R251D, R251E, R251F, R251G, R251H, R251L, R251M, R251N, R251Q, R251S, R251T, or R251W, T254H, T254K, T254W, or T254Y, K256A, K256E, K256T, or K256V, S317C or S317R, N318AN318F, N318H, N318K, N318Q, or N318R, T320A, T320H, T320M, T320N, or T320P, and/or K321D, K321G, K321I, or the particular permutation of K321T, all all see SEQ ID NO:4.

In certain embodiments, variant of the present invention is not contained in the displacement of the wt amino acid residue of the one or more positions corresponding to N88, A252, A253, N308, A316, S357, T400, R402 and the D403 in SEQ ID NO:4.In certain embodiments, variant of the present invention is not contained in the displacement of the wt amino acid residue of the one or more positions corresponding to N4, G5, T38, N93, G94, I95, Q96, V97, Y230, G255, V315, P319, A322, L354, T355, R356, G359, Y396, A397, Y398, G399 and the Q401 in SEQ ID NO:4.

In certain embodiments, what variant of the present invention was included in corresponding in the following residue of the following position of SEQ ID NO:4 is one or more: the N in position 88 in SEQ ID NO:4, the A in position 252, the A in position 253, the N in position 308, the A in position 316, the S in position 357, T, the R in position 402 and the D in position 403 in position 400.In certain embodiments, variant of the present invention is included in all aforementioned residue of specified location.In certain embodiments, it is one or more that variant of the present invention is included in corresponding in the following residue of the following position of SEQ ID NO:4: the N in position 4 in SEQ ID NO:4, at the G of position 5, at the T of position 38, at the N of position 93, at the G of position 94, at the I of position 95, at the Q of position 96, at the V of position 97, at the Y of position 230, at the G of position 255, at the V of position 315, at the P of position 319, at the A of position 322, at the L of position 354, at position 355T, at the R of position 356, at the G of position 359, at the Y of position 396, at the A of position 397, at the Y of position 398, at the G of position 399, with the Q in position 401.4. in certain embodiments, variant of the present invention is included in all aforementioned residue of specified location.

Corresponding position in other amylase is determined by amino acid alignment.Figure 4 and 5 show the position of described groove in bacstearothermophilus amylase.Homology residue in other amylase is determined by structure alignment or by primary structure comparison, as shown in Figure 6.

Variant walsh fireball bacterium amylase uPWA (SEQ ID NO:5)

Bacillus licheniformis alpha-amylase (LAT; SEQ ID NO:2):

Geobacillus stearothermophilus (Geobacillus stearothermophilus) (being called bacstearothermophilus (Bacillus stearothermophilus) in the past) amylase (SEQ ID NO:3):

Cytophaga species α-amylase (AAF00567.1, GI#6006681; SEQ ID NO:4):

According to the compositions and methods of the invention, the corresponding residue in one or more or another family 13 sample α-amylase in the one or more or aforementioned amino acid residue in the residue in said second structural motif suddenlys change changing under the interactional impact between starch engagement groove and starch bundle.In certain embodiments, single residue mutations is made.In other embodiments, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43 or even 44 residue mutations are made.In certain embodiments, the corresponding residue in one or more or another family 13 sample α-amylase in the one or more or aforementioned amino acid residue in the residue in said second structural motif does not suddenly change clearly, and one or more residue then suddenlys change.In certain embodiments, corresponding residue in one or several or another family 13 sample α-amylase in one or several or aforementioned amino acid residue in residue in said second structural motif does not suddenly change clearly, and one or several residues then suddenly change.

In certain embodiments, sudden change is the radical amino acid replacement be present in parent amylase is different amino-acid residues.In certain embodiments, sudden change is the disappearance of the amino-acid residue be present in parent amylase, thus changes and the amino-acid residue in the interactional groove of starch bundle.In certain embodiments, sudden change is the insertion of the amino-acid residue be present in parent amylase, thus changes and the amino-acid residue in the interactional groove of starch bundle.

In certain embodiments, sudden change strengthens the combination of family 13 sample α-amylase and starch bundle, such as, by the interaction of molecules between the residue in enhancing starch engagement groove and starch bundle.Estimate that restriction amylase spreads and improves persistence by this type of sudden change, but they also strengthen thermostability, pH stability or washing composition stability and reduce Ca-dependent.

In certain embodiments, amylase of the present invention is CAZy family 13 amylase.In certain embodiments, to have Bacillus spec diastatic folding for amylase of the present invention.In certain embodiments, amylase of the present invention is the diastatic variant of Bacillus spec, itself and SEQ ID NO:2 or SEQ ID NO:3 have the amino acid sequence homology/identity of restriction degree, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence homology/identity.In certain embodiments, amylase of the present invention is the diastatic variant of Cytophaga species, itself and SEQ ID NO:4 have the amino acid sequence homology/identity of restriction degree, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence homology/identity.In certain embodiments, amylase of the present invention is Pyrococcus species (Pyrococcus sp.) diastatic variant, itself and SEQ ID NO:1 or SEQ ID NO:5 have the amino acid sequence homology/identity of restriction degree, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence homology/identity.

Except sudden change as herein described, the sudden change that amylase of the present invention describes before also can comprising one or more.The sudden change before described knownly has similar folding and/or have in the amylase of amino acid sequence identity of 60% or higher at least one and bacillus amylase, or gives the sudden change of beneficial characteristics in any amylase being called as " Termamyl sample " so far.

In addition, amylase of the present invention can be included in any amount of conservative amino acid displacement of the position of non-specific mutant.Exemplary conservative amino acid displacement is listed in Table 1.

table 1. conservative amino acid is replaced

Amylase of the present invention can be " precursor ", " jejune " or " total length ", and they comprise signal sequence in these cases, or " ripe ", they lack signal sequence in this case.The mature form of polypeptide is normally the most useful.Unless otherwise stated, numbering amino acid residues mode used herein refers to the mature form of each alpha-amylase polypeptide.Alpha-amylase polypeptide of the present invention also can by brachymemma with remove N end or C hold, as long as gained polypeptide retain amylase activity.

Amylase of the present invention can be " being fitted together to " or " heterozygosis " polypeptide, because it comprises (this chimeric amylase " has been rediscovered " recently as Domain swapping amylase) at least partially of at least partially with the second alpha-amylase polypeptide of the first alpha-amylase polypeptide.Amylase of the present invention can also comprise Heterologous signal sequences, make to follow the tracks of or the epi-position etc. of purifying.Exemplary Heterologous signal sequences is from B. licheniformis amylase (LAT), subtilis (B.subtilis) (AmyE or AprE) and streptomycete (Streptomyces) CelA.

In yet another aspect, the nucleic acid of any described alpha-amylase polypeptide of encoding is provided.The specific alpha-amylase polypeptide of described nucleic acid encodes or there is the amylase of the amino acid sequence identity of given extent with described specific amylase.In one example in which, nucleic acid encoding and the amylase with reference to amylase with at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% homology/identity.Should be appreciated that the degeneracy due to genetic code, the polypeptide that multiple nucleic acid encodes is identical.

Nucleic acid also can have the homology of given extent with the Exemplary polynucleotide of coding for alpha-alpha-amylase polypeptide.Such as, nucleic acid can have at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% nucleotide sequence homology with exemplary sequence.And for example, nucleic acid strictly or under very strict condition is being hybridized with exemplary sequence.Described by such condition has in this article, but be also well known in the art.

Nucleic acid encodes " total length " (" fl " or " FL ") amylase (comprising signal sequence), only diastatic mature form (lacking signal sequence) or diastatic clipped form (lacking N end or the C end of mature form).

Coding for alpha-diastatic nucleic acid can be suitable for effectively being connected with various promotor and regulon in express alpha-diastatic carrier in host cell.Exemplary promoters is from B. licheniformis amylase (LAT), subtilis (AmyE or AprE) and streptomycete CelA.This nucleic acid also can be connected with other encoding sequences, such as, in order to encoding chimera polypeptide.

5. for the preparation of the method for Variant Amylase

An aspect of the compositions and methods of the invention prepares the method for Variant Amylase.In this way, the structural features in amylase molecules is introduced in sudden change, acts on needed for relevant to starch to cause.Sudden change can use the molecular biotechnology known to carry out, and the Variant Amylase of gained can use standard method to express as the polypeptide of secretion.Such as, production and purifying are known in the art from genus bacillus (Bacillus) method of protein be secreted into substratum, are also known in the art for the production of diastatic Suitable host cells.Open hereinafter for the production of diastatic illustrative methods.

5.1. for generation of diastatic materials and methods

Use and usually the expression vector comprising control sequence is carried out express polypeptide, described control sequence comprises suitable promotor, operon, ribosome bind site, translation initiation signal, and optionally comprises repressor gene or multiple activated gene.The commercially available acquisition of a large amount of carriers, for recombinant DNA method, and the selection of carrier will depend on that it is about to the host cell introduced wherein usually.Described carrier can be autonomously replicationg vector, and namely as the carrier that extrachromosomal entity exists, it copies and does not rely on chromosome duplication, such as, and plasmid, phage or extra-chromosomal element, minichromosome or artificial chromosome.Alternatively, carrier can be integrated into when it is introduced in the host cell of separation in host cell gene group and with integrate the carrier copied together with its karyomit(e).To be selected by microbiotic or other selective pressures (regulatory gene as required) drive or the increased construct that driven by the supplementing of dosage effect to required metabolic pathway gene by using, the gene of described integration of also can increasing is to produce multiple copies of gene described in karyomit(e).

In the carrier, DNA sequence dna should be effectively connected to suitable promoter sequence.Described promotor can be any DNA sequence dna showing transcriptional activity in the host cell selected, and can be derived from the gene of the protein of coding and host cell homology or allos.For guiding transcribing of the DNA sequence dna of encode starch enzyme, especially the Exemplary promoters of transcribing in host bacterium is the promotor of intestinal bacteria (E.coli) lactose operon, streptomyces coelicolor (Streptomyces coelicolor) agarase gene dagA or celA promotor, the promotor of bacillus licheniformis alpha-amylase gene (amyL), the promotor of bacstearothermophilus (Bacillus stearothermophilus) maltogenic amylase gene (amyM), the promotor of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) α-amylase (amyQ), the promotor etc. of subtilis (Bacillus subtilis) xylA and xylB gene.For transcribing in fungal host, the example of available promotor is those promotors derived from the gene of encoding A (Aspergillusoryzae) TAKA amylase, Rhizomucor miehei (Rhizomucor miehei) aspartate protease, aspergillus niger (Aspergillus niger) neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger glucoamylase, Palatase, line protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans (A.nidulans) acetamidase.When expressing the gene of encode starch enzyme in such as colibacillary bacterial species, such as can select suitable promotor from phage promoter, described phage promoter comprises T7 promotor and lambda particles phage promotor.The example being applicable to the promotor expressed in yeast species includes but not limited to the Gal 1 of yeast saccharomyces cerevisiae (Saccharomycescerevisiae) and AOX1 or the AOX2 promotor of Gal 10 promotor and pichia pastoris phaff (Pichia pastorisor).For the expression in Trichodermareesei (Trichoderma reesei), CBHII (cellobiohydrolase II) promotor can be used.

Expression vector also can comprise and the suitable transcription terminator that coding for alpha-diastatic DNA sequence dna is effectively connected and the Polyadenylation sequences in eukaryote.Terminator sequence can be derived from the source identical with promotor aptly with Polyadenylation sequences.

Carrier also can comprise the DNA sequence dna that carrier can be copied in host cell.The example of this kind of sequence is the replication orgin of plasmid pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

Carrier also can comprise selectable marker, as its product can supply the gene of the defect in the host cell of separation, as the dal gene from subtilis or Bacillus licheniformis, or give the gene of antibiotics resistance (as penbritin, kantlex, paraxin or tetracyclin resistance).In addition, carrier can comprise Aspergillus (Aspergillus) selective marker, as amdS, argB, niaD and xxsC (producing the mark of hygromycin resistance), or realizes selecting by such as cotransformation known in the art.See such as pct international patent application WO 91/17243.

As described above, although cell inner expression or solid state fermentation can be favourable in some respects, such as, when some bacterium or fungi are used as host cell, an aspect of described composition and method contemplates to be expressed α-amylase into substratum.

Usually, " total length ", " maturation " or " precursor " amylase are included in aminoterminal signal sequence, and it allows to be secreted in substratum.If needed, this signal peptide can be replaced by different sequence, this displacement by the DNA sequence dna of coding corresponding signal polypeptide and realizing easily.

Expression vector comprises the component of cloning vector usually, such as, in the host living beings selected, allows the element of carrier self-replicating and for selecting one or more phenotype detectable labels of object.Expression vector comprises the control nucleotide sequence of such as promotor, operon, ribosome bind site, translation initiation signal and optional repressor gene or one or more activated gene usually.In addition, expression vector can comprise coding can by amylase target to host cell organelle (as peroxysome) or target to the sequence of the aminoacid sequence of particular host cell compartment.This target sequence includes but not limited to sequence SKL.For the expression under control sequence guiding, diastatic nucleotide sequence is effectively connected with control sequence in suitable mode with regard to expressing.

For connecting the operation of the DNA construct of encode starch enzyme, promotor, terminator and other elements respectively, with must the operation of suitable carrier of information be well known to the skilled person (see such as Sambrook et al. for their being inserted containing copying, MOLECULAR CLONING:ALABORATORY MANUAL, 2 ^nded., Cold Spring Harbor, 1989, and 3 ^rded., 2001 (people such as Sambrook, " molecular cloning: laboratory manual ", the 2nd edition, CSH Press, 1989, and the 3rd edition, calendar year 2001)).

The cell comprising the separation of DNA construct or expression vector is advantageously used as host cell in diastatic recombinant production.The DNA construct of available code enzyme, is integrated in host chromosome carrys out transformant conveniently by by described DNA construct (with one or more copy).It has been generally acknowledged that this integration is favourable, because DNA sequence dna more likely stably maintains in cell.According to conventional methods, such as, by homology or heterologous recombination, DNA construct can be realized and is integrated in host chromosome.Alternatively, the above-mentioned expression vector that available with dissimilar host cell is combined transforms cell.

The example of suitable bacterial host organisms is gram positive bacterium species, as Bacillaceae (Bacillaceae) (comprises subtilis, Bacillus licheniformis, bacillus lentus (Bacilluslentus), bacillus brevis (Bacillus brevis), Geobacillus stearothermophilus (Geobacillusstearothermophilus) (being called bacstearothermophilus) before, Alkaliphilic bacillus (Bacillusalkalophilus), bacillus amyloliquefaciens, Bacillus coagulans (Bacillus coagulans), bacillus lautus (Bacillus lautus), bacillus megaterium (Bacillus megaterium) and bacillus thuringiensis (Bacillus thuringiensis), streptomycete species, such as mouse ash streptomycete (Streptomycesmurinus), milk-acid bacteria species, comprise lactococcus (Lactococcus) species as Lactococcus lactis (Lactococcus lactis), lactobacillus (Lactobacillus) species, comprise lactobacillus reuteri (Lactobacillus reuteri), leuconos toc (Leuconostoc) species, Pediococcus (Pediococcus) species, and streptococcus (Streptococcus) species.Alternatively, the bacterial strain of the gram negative bacterium species belonging to enterobacteriaceae (Enterobacteriaceae) (comprising intestinal bacteria) or belong to pseudomonadaceae (Pseudomonadaceae) can be selected as host living beings.

Suitable YEAST HOST ORGANISMS can be selected from the yeast species that biotechnology is correlated with, described yeast species is such as but not limited to such as pichia spp species (Pichia sp.), debaryomyces hansenii species (Hansenula sp.), or kluyveromyces spp (Kluyveromyces), Yarrowinia, the yeast species of Schizosaccharomyces (Schizosaccharomyces) species or the species (comprising yeast saccharomyces cerevisiae) of yeast belong (Saccharomyces), or belong to species such as schizosaccharomyces pombe (S.pombe) species of Schizosaccharomyces.The bacterial strain of methylotrophic yeast species pichia pastoris phaff can be used as host living beings.Or host living beings can be debaryomyces hansenii species.Host living beings suitable in filamentous fungus comprises the species of Aspergillus, as aspergillus niger, aspergillus oryzae, Tabin aspergillus (Aspergillus tubigensis), Aspergillus awamori (Aspergillus awamori) or Aspergillus nidulans.Alternatively, the bacterial strain of Fusarium (Fusarium) species, as Fusarium oxysporum (Fusarium oxysporum), or the bacterial strain of Rhizomucor (Rhizomucor) species, such as Rhizomucor miehei can be used as host living beings.Other suitable bacterial strains comprise thermophilic fungus and belong to (Thermomyces) and Mucor (Mucor) species.In addition, Trichodermareesei can be used as host.To transform the appropriate method of Aspergillus host cell and comprise (such as) described in EP 238023.

In yet another aspect, provide the method for production α-amylase, described method cultivates above-mentioned host cell under being included in the condition being conducive to enzyme production, and reclaims enzyme from described cell and/or substratum.

Substratum for culturing cell can be that any being suitable for cultivates the host cell considered and the conventional medium obtaining amylase expression.Suitable substratum and nutrient media components available from commercial supplier or can be prepared according to the formula (formula such as, as described in the catalogue at American type culture collection (American TypeCulture Collection)) announced.

In one aspect, whole beer preparation is used for from the enzyme of host cell secretes.In the method for the invention, use any cultural method causing α-amylase to be expressed known in the art, the preparation of the whole beer of the consumption of recombinant microorganism can be realized.Therefore, fermentation can be interpreted as and be included in suitable culture medium and the in vitro shake-flask culture that carries out or the small-scale in industrial fermentation tank or large scale fermentation (comprise continuously ferment, batch fermentation, fed-batch fermentation or solid state fermentation) under the condition allowing amylase to express or to be separated.Term " whole beer of consumption " is defined as the non-classification contents thing of fermented material in this article, comprises substratum, extracellular protein (such as enzyme) and cellular biomass.Should be appreciated that term " whole beer of consumption " also covers the cellular biomass using method well known in the art to make its cracking or saturatingization.

From substratum, the enzyme of secretion from host cell is reclaimed easily by known method, described method comprises by centrifugal or filtration isolated cell from substratum, and by means of the protein component in the salt precipitation substratum of such as ammonium sulfate and so on, use the chromatographic process of such as ion exchange chromatography, affinity chromatography etc. and so on subsequently.

The aspect polynucleotide contemplated in carrier are effectively connected to the control sequence that can be provided the expression of encoding sequence by host cell, and namely carrier is expression vector.Control sequence can such as be modified by adding other transcriptional regulatory elements, thus the response of the transcriptional level that control sequence is guided to transcription regulaton factor is sensitiveer.Control sequence especially can comprise promotor.

Host cell can be cultivated under the conditions suitable allowing amylase to express.The expression of enzyme can be composing type, makes them can continuous seepage; Can be maybe induction type, thus need stimulator to cause expression.When inducible expression, such as, protein generation can be caused by adding inductive substance (such as dexamethasone or IPTG or Sopharose) in substratum when needed.Also can in vitro cell-free system (as TNT ^tM(Promega) rabbit reticulocyte system) middle recombinant production polypeptide.

Express diastatic host also can be applicable to described host substratum in, cultivate under aerobic conditions.The combination that can provide vibration or stir and ventilate, produces at the temperature such as about 25 DEG C to about 75 DEG C (such as 30 DEG C to 45 DEG C) being applicable to described host, and this depends on the needs of host and produces required diastatic needs.Can cultivate about 12 to about 100 hours or longer (and any time value therebetween, as 24 to 72 hours).Usually, the pH of cultivation and fermentation liquid is about 5.5 to about 8.0, and this also depends on the culture condition needed for the host relevant to diastatic production.

5.2. for the materials and methods of protein purification

Fermentation, isolation and identification technology are well known in the art, and ordinary method can be used to prepare the solution of concentrated amylase-containing polypeptide.

After fermentation, obtain fermented liquid, and remove the solid substance (comprising residual fermentation raw material) of microorganism cells and various suspension to obtain amylase solution by conventional isolation techniques.Usual use is filtered, centrifugal, micro-filtration, rotating drum vacuum filtration, ultrafiltration, centrifugal and ultrafiltration that is that carry out subsequently, extraction or chromatography etc.

The preferably concentrated α-amylase polypeptide solution that contains is to optimize the rate of recovery.Unconcentrated solution is used to need to increase incubative time to collect the enzyme precipitation of purifying.

Conventional concentration technique is used to concentrate containing enzyme solution until enzyme content needed for obtaining.Concentrating containing enzyme solution is realized herein by any technology discussed.The illustrative methods of purifying includes but not limited to rotary vacuum filtration and/or ultrafiltration.

Enzyme solution is condensed into concentrated enzyme solutions until the enzymic activity of concentrated amylase-containing polypeptide solution is in required level.

Such as precipitation agent (such as metal halide precipitate agent) can be used to concentrate.Metal halide precipitate agent includes but not limited to: two or more blend in alkali metal chloride, alkali metal bromide and these metal halides.Exemplary metal halide to comprise in sodium-chlor, Repone K, Sodium Bromide, Potassium Bromide and these metal halides two or more blend.Metal halide precipitate agent sodium-chlor also can be used as sanitas.

Metal halide precipitate agent uses with the amount that effectively can precipitate α-amylase polypeptide.That after conventionally test, selects the metal halide that can effectively cause enzyme to precipitate has effective amount and optimal dose at least, and the deposition condition of maximum recovery (comprising incubative time, pH, temperature and enzyme concn), will be apparent to those skilled in the art.

Generally speaking, add the metal halide at least about 5%w/v (weight/volume) to about 25%w/v to concentrated enzyme solution, normally at least 8%w/v.Generally speaking, add the metal halide being no more than about 25%w/v to concentrated enzyme solution, be normally no more than about 20%w/v.Except other aspects, the optimal concentration of metal halide precipitate agent will depend on the character of concrete alpha-amylase polypeptide and its concentration in concentrated enzyme solution.

Affect enzyme precipitation another can selection scheme be use organic compound.Exemplary organic compound precipitation agent comprises: two or more blend in an alkali metal salt of 4-HBA, 4-HBA, the alkyl ester of 4-HBA and these organic compound.The interpolation of described organic compound precipitation agent can before the agent of interpolation metal halide precipitate, with its simultaneously or occurring thereafter, and the interpolation of two kinds of precipitation agents (organic compound and metal halide) can in succession be carried out or carry out simultaneously.

Usually, organic precipitant to be selected from an alkali metal salt (as sodium or sylvite) of 4-HBA and the straight or branched alkyl ester (wherein alkyl contains 1 to 12 carbon atom) of 4-HBA and these organic compound the blend of two or more.Organic compound precipitation agent can be two or more blend in the straight or branched alkyl ester (wherein alkyl contains 1 to 10 carbon atom) of (such as) 4-HBA and these organic compound.Exemplary organic compound is two or more blend in the straight chained alkyl ester (wherein alkyl contains 1 to 6 carbon atom) of 4-HBA and these organic compound.Also the blend of two or more can be used in the propyl ester of the methyl esters of 4-HBA, 4-HBA, the butyl ester of 4-HBA, the ethyl ester of 4-HBA and these organic compound.Other organic compound also includes but not limited to 4-HBA methyl esters (methyl p-hydroxybenzoate by name) and 4-HBA propyl ester (propylparaben by name), and they are also all amylase sanitass.Relevant further description, see such as U.S. Patent No. 5,281,526.

With regard to pH, temperature, alpha-amylase polypeptide concentration, precipitant concentration and incubative time, be added with the advantage that organic compounds precipitation agent provides deposition condition high degree of flexibility.

Organic compound precipitation agent uses with the amount effectively improving enzyme precipitation by metal halide precipitate agent.According to the disclosure, that after conventionally test, selects organic compound precipitation agent has effective amount and optimal dose at least, and the deposition condition of maximum recovery (comprising incubative time, pH, temperature and enzyme concn), will be apparent to those skilled in the art.

Generally speaking, the organic compound precipitation agent at least about 0.01%w/v is added, normally at least about 0.02%w/v to concentrated enzyme solutions.Generally speaking, add the organic compound precipitation agent being no more than about 0.3%w/v to concentrated enzyme solutions, be normally no more than about 0.2%w/v.

Can regulate the pH of the condensing peptide solution containing metal halide precipitate agent and organic compound precipitation agent, described pH must depend on enzyme to be purified.Usually, by the level near pH regulator to amylase iso-electric point.PH can be regulated within the scope of the pH being extremely about 2.5pH unit lower than iso-electric point (pI) about 2.5pH unit higher than iso-electric point.

The incubative time needed for enzyme throw out obtaining purifying depends on the character of concrete enzyme, enzyme concn and concrete precipitation agent and concentration thereof.Generally speaking, the time of enzyme can effectively be precipitated between about 1 to about 30 hour; Usually about 25 hours are no more than.When having organic compounds precipitation agent, incubative time can also reduce to lower than about 10 hours, in most of the cases or even about 6 hours.

Generally speaking, the temperature between incubation period is between about 4 DEG C to about 50 DEG C.Usually, between about 10 DEG C to about 45 DEG C (e.g., between about 20 DEG C to about 40 DEG C) temperature under carry out described method.Optimum temps for induced precipitation changes according to solution condition and enzyme or precipitation agent used.

The sedimentary total yield of enzyme of purifying and the efficiency of the practice of described method is improved by stirring the solution comprising enzyme, the metal halide of interpolation and the organic compound of interpolation.During interpolation metal halide and organic compound, and carry out whipping step in incubation period process subsequently.Suitable stirring means comprises mechanical stirring or vibration, sharp draft or any similar technology.

After incubation period, by the enzyme of purifying and the pigment dissociated and other magazins' layout, and by conventional isolation techniques (such as filter, centrifugal, micro-filtration, rotary vacuum filtration, ultrafiltration, press filtration, cross-film micro-filtration, cross-flow membrane micro-filtration etc.) collect.Can be further purified the enzyme of purifying is sedimentary by washing throw out acquisition with water.Such as, with the water containing metal halide precipitate agent or the enzyme throw out washing purifying with the water containing metal halide and organic compound precipitation agent.

During the fermentation, α-amylase polypeptide is collected in cultivation and fermentation liquid.For required diastatic abstraction and purification, carry out centrifugal to nutrient solution or filter to remove cell, and the cell free fluid of gained is used for enzyme purification.In one embodiment, the ammonium sulfate of about 70% saturation ratio is used to saltout to acellular nutrient solution; Then the fraction of 70% saturation ratio precipitation is dissolved in damping fluid, then is applied on the post of such as Sephadex G-100 post and so on, and wash-out is to reclaim enzymic activity fraction.In order to further purifying, the ordinary method of such as ion-exchange chromatography can be used.

Enzyme after purifying can be used for clothes washing and cleaning applications.Such as, they may be used in laundry detergent and Scouring agent.They can be made the finished product of liquid (solution, slurries) or solid (particle, powder) form.

The example more specifically of purifying is at Sumitani, J.et al. (2000) " New type of starch-binding domain:the direct repeat motif in the C-terminal region of Bacillus sp.195 α-amylase contributes to starch binding and raw starch degrading, " Biochem.J.350:477-484 (Sumitani, J. people is waited, 2000, " novel starch binding domain: the motif of repetition in the same way in the C end regions of Bacillus spec No. 195 α-amylase contributes to starch and combines and uncooked amylum degraded ", " journal of biological chemistry ", 350th volume, 477-484 page) in have described and carry out brief overview here.With (the NH of 80% saturation ratio ₄) ₂sO ₄process the enzyme obtained from 4 liters of muta lead mycillins (Streptomyces lividans) TK24 culture supernatant.By 10, under 000 × g, (20 minutes and 4 DEG C) are centrifugal and reclaim throw out, and are again dissolved in it containing 5mM CaCl ₂20mM Tris/HCl damping fluid (pH 7.0) in.Then with identical damping fluid, the precipitation of dissolving is dialysed.Then the sample of dialysing is applied to previously with containing 5mM CaCl ₂the Sephacryl S-200 post that balances of 20mM Tris/HCl damping fluid (pH 7.0) on, then use same buffer with the linear rate of flow wash-out of 7mL/h.Collect the fraction from post, then assess the activity that it judges by enzyme assay and SDS-PAGE.Be further purified protein as follows.ToyopearlHW55 post (Dong Cao Life Sciences of Montgomery city of Pennsylvania (Tosoh Bioscience, Montgomeryville, PA); Cat. numbering 19812) with containing 5mM CaCl ₂with 1.5M (NH ₄) ₂sO ₄20mM Tris/HCl damping fluid (pH 7.0) balance.With containing 5mM CaCl ₂20mM Tris/HCL damping fluid (pH 7.0) linear gradient be the (NH of 1.5 to 0M ₄) ₂sO ₄wash-out enzyme.Collect active fraction, and with (the NH of 80% saturation ratio ₄) ₂sO ₄enzyme is precipitated.As mentioned above throw out is reclaimed, again dissolve and dialyse.Then the sample after dialysis is applied to Mono Q HR5/5 post (peace Pharmacia biotech company (Amersham Pharmacia); Cat. numbering 17-5167-01), described post is previously with containing 5mM CaCl ₂20mM Tris/HCl damping fluid (pH 7.0) balance with the flow velocity of 60mL/h.Collect active fraction, and added to 1.5M (NH ₄) ₂sO ₄in solution.Making organized enzyme fraction chromatography again on Toyopearl HW55 post as previously mentioned, obtaining the homogeneous enzyme as determined by SDS-PAGE.See Sumitani, J.et al. (2000) Biochem.J.350:477-484 (people such as Sumitani, J., 2000, " journal of biological chemistry ", the 350th volume, 477-484 page), to understand the generality discussion of its method and change.

For production-scale recovery, as above can be described through in general manner with polymer flocculation removing cell and partial purification is carried out to alpha-amylase polypeptide.Alternatively, available film and equipment can be used by micro-filtration, next undertaken concentrated by ultrafiltration and purifying is carried out to enzyme.But, for some application, without the need to carrying out purifying to enzyme, and can dissolve whole beer culture without the need to processing further and use.Then enzyme can be processed into (such as) particle.

6. comprise the composition of variant alpha amylase

Present invention also offers the composition comprising alpha-amylase variants described in one or more.Such composition comprises the zymin of such as enzyme enriched material, enzyme blend, purifying, partially purified enzyme product, the fermented liquid product of clarification, whole beer product, foodstuff additive and cleaning product.Described composition can multiple physical form provide, and comprises liquid, slurries, gel, muffin, powder, particle etc.Described composition can multiple known or available mode freeze-drying, concentrated, freezing, spraying dry or otherwise process.Described composition can provide for some commercial applications by normal size, or carries out customization packaging, or provides in the bulk container of any type.

Described composition can also comprise the preparation composition of any amount or be combined with the preparation composition of any amount, described preparation composition such as buffer reagent, salt, sequestrant, sanitas, biocide, polymkeric substance, weighting agent etc.Especially, when composition is cleaning compositions, they can also comprise such as tensio-active agent, oxygenant, sequestrant or other sanitising agents.Exemplary composition is laundry detergent and dishwashing detergent, comprises automatic dishwasher washing composition.Described composition can also comprise one or more other polypeptide or the polypeptide other with one or more is combined.One or more other polypeptide described can comprise at least one enzyme.Other enzyme includes but not limited to other α-amylase, beta-amylase, glucoamylase, isoamylase, isomerase, phytase, proteolytic enzyme, cellulase, ligninase, hemicellulase, lipase, Phospholipid hydrolase and at.

In certain embodiments, the enzyme blend comprising one or more the composition in Variant Amylase of the present invention or also comprise one or more other enzymes can be used for liquefying starch.In certain embodiments, described composition can be used for being conducive to removing starch from yarn fabric, paper, glass, plastics, metal, canvas, porcelain and other surfaces.In certain embodiments, described composition is produced or is mixed with foodstuff additive or the processing aid as being applicable to food technology.Described composition starch be the form of starch bundle at least partially time especially effective.

8. using method

Present invention also offers the method for the composition using described variant alpha amylase or comprise this type of variant.In certain embodiments, the method for the complex carbohydrates that is used for by described variant alpha amylase liquefying, such as liquefying starch slurries, especially when farinaceous size contains starch bundle.Described method comprises the slurries that preparation comprises complex carbohydrates substantially, slurries are heated to the acceptable temperature that liquefies, the composition comprising at least one alpha-amylase variants as herein provided is added in slurries, and by slurries and composition incubation for some time at the temperature of complex carbohydrates that is enough to liquefy.As used herein, " liquefaction " does not mean that each available substrate bonding of cracking, but represent and be hydrolyzed complex carbohydrates at least in part, as the measurability by final viscosity reduces, the increase of slurries DE, the release of low DP fragment/product or reduction group, dextrin or α-maltose unit increase another measure confirm.

The temperature of liquefaction in room temperature in the scope more than 100 DEG C, but can be more preferably about 50 DEG C to about 95 DEG C.Liquefaction may need the Complex Temperature curve using time to time change, and such as reaction can start at low temperatures, and rises to the outlet temperature of expectation by methods known in the art.Also can after specifying the long time, or temperature is reduced after reaching the expectation terminal that viscosity, DE value or another liquefaction measures.Therefore technician will know, described method does not need to limit the specific constant temperature specific time, as long as amylase activity can work at provided temperature and condition.Except the whether existence of inhibitor etc., other conditions of impact activity comprise pH and calcium ion concn.

Slurries can comprise the starch being about 20-40% by dry weight basis, and such as slurry package is containing the starch of about 30% to about 36% or 37.5%.The starch of less amount can be used, but usually uneconomical.Peak viscosity and correlative factor (such as mixing required energy input) can limit the maximum of the starch be used in slurries.Technician is preparing the actual Consideration in farinaceous size by knowing.Technician will know, viscosity reduces more, and starch liquefacation must be more thorough, and dextrin produces more (or the DE of gained liquefying starch is higher).Therefore in one embodiment, the wild-type enzyme liquefaction be derived from respect to variant or the peak viscosity of comparable slurries by processing with enzyme preparation commercially available at present, peak viscosity reduces at least 10,20,25,30,40 or even 50% or higher.

In one embodiment, liquefaction is that a part for fermentation is to produce such as foods prods, foodstuff additive, fuel or fuel dope etc.In certain embodiments, fermentation produces fuel or fuel dope, such as alcohol, such as ethanol, butanols or another lower alcohol.The present invention clearly provides the method using variant alpha amylase as herein described, composition or enzyme blend to produce ethanol.According to these class methods, after interpolation variant enzyme, fermented by the liquefying starch slurries of gained with one or more organisms, described organism can be suitable for fermenting the condition that produces ethanol and produce ethanol under the time.Alternatively, there is Variant Amylase and organism, as with regard to simultaneous saccharification and fermentation (SSF) simultaneously.

Also provide clean surface to remove the method for undesired or unexpected starch residue herein, especially when starch be starch bundle form at least partially time.Said method comprising the steps of: the surface with starch residue to be removed is provided, make described surface at enough temperature with the condition making to remove starch residue, contact for some time with the composition comprising one or more variant alpha amylase.Described surface can on any material; Such as, they can on plate, flat board, glass etc., or they can on clothes or fabric.It also can be such as counter top or work top, or the commercially available container of any type that must regularly or regularly clean.

The method using alpha-amylase variants process weaving material as herein described is also provided herein.Be known in the art by the method for amylase process weaving material (such as fabric) and contain the process being called " destarch ".Described method comprises the composition making weaving material and the liquid comprising alpha-amylase variants or comprise variation and contacts.In one embodiment, weaving material is fabric or yarn fabric.In another embodiment, under stress with described liquid treatment weaving material.Described liquid is generally aqueous solution.Alpha-amylase variants provided herein can be used alone or uses with destarch weaving material, such as fabric together with other destarch chemical reagent (such as washing composition and/or destarch enzyme), comprises cotton and cotton-containing fabrics.

For all objects, above-cited all reference are incorporated herein by reference all in full.Hereafter provide working example to further describe and to explain some aspect of the variant of α-amylase, and therefore should not be considered as restrictive.

example

the purifying of example 1:uPWA

Derived from the variant alpha amylase uPWA (U.S. Patent No. 7,273, the SEQ ID NO:2 in 740) of walsh fireball bacterium purchased from Verenium company (California, USA San Diego (SanDiego, CA, USA)).UPWA comprises 58 amino-acid substitutions relative to wild-type walsh fireball bacterium amylase PWA.The aminoacid sequence of uPWA and PWA is respectively as shown in hereafter SEQ ID NO:5 and SEQ ID NO:1.

Variant walsh fireball bacterium amylase uPWA (SEQ ID NO:5)

Wild-type walsh fireball bacterium amylase (SEQ ID NO:1)

By uPWA by hydrophobic interaction chromatography purifying transferring to by 50mM glycine (pH10) and 2mM CaCl ₂in the high pH damping fluid of composition, then mix 20 minutes.Ammonium sulfate is added to the ultimate density reaching 1M, and makes solution remix 30 minutes, be then applied on the equilibrated 30mL phenyl sepharose post of use same buffer.By the identical buffer solution of pillar, until reach stable baseline.Apply the gradient of 300mL to reach 50mM glycine (pH 10), 2mMCaCl ₂, then in identical damping fluid, carry out 300mL washing.Finally, by protein wash-out in 50mM glycine (pH 10), 2mM calcium chloride, 40% propylene glycol.

The cut of mixing is concentrated in Vivaspin concentrating instrument (MWCO 10,000) final protein concentration of 20mg/mL.By the elution buffer of the protein storage of purifying at 4 DEG C (50mM glycine (pH 10), 2mM CaCl ₂, 40% propylene glycol) in.By the spectrodensitometry concentration under 280nm, and SDS-PAGE is used for evaluating purity.Before crystallization, protein storage damping fluid Vivaspin concentrating instrument (MWCO 10,000) is exchanged into water, and final protein concentration is adjusted to 10mg/ml.

the crystallization of example 2:uPWA

In Cryschem plate (Hampton research company (Hampton Research)), at room temperature use 24 holes seats to drip carry out crystallization experiment.Commercially available screening scheme (Hampton company (Hampton) and Kai Jie company (Qiagen)) is used to carry out initial screening.Be mixed to form to sit with the stock solution (reservoir solution) of 2 μ l protein examples and equivalent and drip, then leave standstill with for 250 μ l stock solution balances.Crystal occurred after 5-7 days, and wherein stock solution is made up of 0.1M Tris-HCl pH 8.5 and 20% (v/v) ethanol or 0.2M NaCl, 0.1M HEPES pH 7.5 and 10% (v/v) Virahol.Before X-ray diffraction analysis, by dripping middle interpolation glucose crystal grain (making it to dissolve) to seat by crystal cryoprotection.Crystal is contained in nylon jacket, and quenching in liquid nitrogen.

Be used in the synchrotron radiation image data of Stamford synchrotron radiation light source (Stanford Synchrotron Radiation Lightsource) (SSRL light beam line BL12-2).Acquire complete data set, whole merga pass XDS with resolving power convergent-divergent (Kabsch, W. (1993) J.Appl.Crystallogr.26:795-800 (Kabsch, W., " application crystallography magazine ", the 26th volume, 795-800 page in 1993)).

example 3.uPWA structure determination and refining

Phaser is used to be determined the structure (McCoy of uPWA by molecular replacement technique, A.J.et al. (2007) J.Appl.Crystallogr.40:658-674 (McCoy, A.J. people is waited, 2007, " application crystallography magazine ", 40th volume, 658-674 page)), wherein by wild-type walsh fireball bacterium amylase (PDB fetcher code: 1MWO; Linden, A.et al. (2003) J.Biol.Chem.278:9875-9884 (people such as Linden, A., " journal of biological chemistry ", the 278th volume, 9875-9884 page in 2003)) structure be used as search model.Use PHENIX (Adams, P.D.etal. (2002) Acta Crystallogr.D Biol.Crystallogr.58:1948-1954 (Adams, P.D. people is waited, 2002, " crystallization journal D collects: organic crystallization ", 58th volume, 1948-1954 page)) and COOT (Emsley, P.and Cowtan, K. (2004) Acta Crystallogr.D Biol.Crystallogr.60:2126-2132 (Emsley, and Cowtan P., K., 2004, " crystallization journal D collects: organic crystallization ", 60th volume, 2126-2132 page)) carry out model refine, map interpretation and model construction.Model refine statistics is listed in Table 1.

table 1. model refine is added up

¹r _work(%)=100 × ∑ _hkl│ Fobs-Fcalc │/∑ _hklfobs, wherein Fobs and Fcalc be respectively observation with calculate structure factor. ²r _freely(%) use 5% reflectance data sample of the Stochastic choice saved from refine and calculate. ³the root-mean-square deviation of desirable bond distance and bond angle.

The mensuration of structure is reached resolving power.Final mask covers ion (4 Ca of the residue 1 to 435 of polypeptide, 2 sucrose moleculess, bonding ²⁺, 1 Zn ²⁺, 1 Tris ⁺, 1 SO ₄ ^2-), 191 water moleculess and 1 beta-cyclodextrin molecule, and with resolving power obtain the R of 16.5% and 20.8% respectively _workand R _freelyvalue, and the wherein residue of 94.2% (Chen in the best region of ramachandran map Ramachandran, V.B.et al. (2010) Acta Crystallogr.Sect.D-Biol.Crystallogr.66:12-21 (Chen, V.B. people is waited, calendar year 2001, " crystallization journal D collects: organic crystallization ", the 66th volume, 12-21 page)).

UPWA configuration shows has the typical case folding (Fig. 1) of CAZy glycosyl hydrolase 13 family in characteristic A, B and C-structure territory.Secondary structure is shown in Figure 2.Although there are 58 sudden changes relative to wild-type walsh fireball bacterium amylase, structure (wtPWA, PDB fetcher code: 1MWO described before overall structure is similar to; Linden, A.et al. (2003) J.Biol.Chem.278:9875-9884 (people such as Linden, A., " journal of biological chemistry ", the 278th volume, 9875-9884 page in 2003)), at C ^αroot-mean-square deviation on backbone locations is center A structural domain (residue 1-109 and residue 170-340) maintains characteristic TIM barrel fold and has active-site residues Asp ²⁸⁹, Glu ²²²and Asp ¹⁹⁸(Banner, D.W.et al. (1975) Nature 255:609-614 (people such as Banner, D.W., " nature ", the 255th volume, 609-614 page in 1975)).Relatively little B structural domain constitutes a part for substrate binding cleft, and there is Ca, Zn-metal center (Linden, A.et al. (2003) J.Biol.Chem.278:9875-9884 (Linden, A. people is waited, 2003, " journal of biological chemistry ", 278th volume, 9875-9884 page)).C holds C-structure territory (residue 341-435) to be made up of the antiparallel β sheet (β-sheet) of eight chains comprising classical Greece key motif (Greek key motif).Except the Ca in the metal center of uniqueness ²⁺and Zn ²⁺outside ion, three Ca are found again ²⁺site: two in A structural domain, one in C-structure territory, all Mg all corresponding to site or the wtPWA α-acarbose (PDB fetcher code: 1MXO) reported for the wtPWA (PDB fetcher code is respectively 1MXD and 1MWO) had He not there is α-acarbose before ²⁺site.

Observed strong anomalous peak at Zn binding site, and do not observe peak at any other binding site.Surprisingly, the cyclodextrin (Fig. 1) of combining closely has been found at the N end of A structural domain.Described binding site is by two rings of the minor spiral before Article 1 beta chain, spiral 6/ chain 7 and spiral 7/ chain 8 and the ring formation being connected A and C-structure territory.A part for cyclodextrin binding site is overlapping with the α observed in wild type peptide-acarbose binding site (Ac-II).Sulfate ion is positioned at cyclodextrin Ring current distribution.

In addition, sucrose molecules is combined between spiral 4/ chain 5 and two rings of spiral 5/ chain 6, near cyclodextrin.Second sucrose molecules has been found near the spiral 3b and C end portion of structural domain B, overlapping with the TEG monomethyl ether binding site of the wild type peptide (PDB fetcher code: 1MXD) with α-acarbose.

58 amino-acid substitutions in uPWA all do not relate to the residue directly contacted with metal ion or other parts; But C153A, C154G, E156S and H168D are near Ca, Zn-metal center, and D358Y, S359G, R360D, R361K and P372S are near the cyclodextrin combined.Major part displacement is positioned on the surface of enzyme.

associating between example 4:SEL victor puts with slot

Prepare the site evaluation libraries (SEL) of 488 complete positions screening at starch coupling collar (R178+ Δ G179; With underscore in SEQ ID NO:4) in there is Cytophaga species amylase (AAF00567.1, the GI#6006681 of disappearance; SEQ ID NO:4) variant.

Cytophaga species α-amylase (AAF00567.1, GI#6006681; SEQ ID NO:4):

This type of SEL is well known in the art, and is usually used in evaluating each displacement or the effect of mutation combination in polypeptide.Based on the ability of its hydrolysed corn starch, to the Performance Score of variant Cytophage alpha-amylase polypeptide.Active performance index (PI) is called by for the expression activity of normalized variant and the ratio of the diastatic activity of wild-type Cytophaga species.SEL " victor " variant that is active PI>1, shows the increased activity to W-Gum thoroughly.But, cause the sudden change of the active <1 of PI usually can suddenly change with other and combine and performance advantage is provided.The expression level of variant and the ratio of the diastatic expression level of wild-type Cytophaga species are called expresses PI.When analyzing SEL data, usually it is appreciated that will derive from expression PI and get rid of, to avoid the error caused because measuring low-level enzymic activity lower than the data of the variant of pre-selected threshold (such as <0.3).

Fig. 6 is histogram, shows the PI distribution of the activity value of all SEL variants of expressing PI>0.3.X-axis represents active PI, and Y-axis represents the quantity of the variant with corresponding active PI.Fig. 7 is histogram, show and only put (namely in slot, position 1,2,3,4,38,88,91,92,93,94,95,96,97,230,251,252,253,254,255,256,308,314,315,316,317,318,319,320,354,355,356,357,358,359,396,397,398,399,400,401,402 and 403, see SEQ ID NO:4) in there is displacement and there is the active PI Distribution value of the SEL variant of the expression PI of >0.3.The distribution of groove positional variant is more partial to Gaussian distribution, and wherein less variant has the active PI of <1.These data show, with indiscriminate (namely, from any position of position 1 to 488 in alpha-amylase polypeptide) sudden change carried out compares, the sudden change of putting in slot more may produce the Variant Amylase of active PI>0.8, >0.9 and >1, and unlikely produces the Variant Amylase of active PI<1, <0.9 and <0.8.

example 5: associating between the activity to amylase substrate is put in slot

Test deriving from the expression of each variant in SEL library of example 4, the activity to amylopectin substrate and the activity to pullulan substrate.The non-branching component of the starch that amylose starch is made up of the glucosyl residue in α-Isosorbide-5-Nitrae bonding.Starch molecule in amylose starch is elongated, and is similar to and is present in the intrafascicular starch molecule of starch.Amylopectin is the branched component of the starch be made up of α-1,6 bonding and α-Isosorbide-5-Nitrae bonding.

The result measured illustrates in figs. 8 and 9.Shown in amino acid position number (Pos) and the wildtype residues (WT) be present on those positions arrange the 1st and the 2nd respectively.Shown in the amino-acid substitution be present on the specified location in tested variant arranges adjacent 20.Grey in cell highlights expression and does not prepare variant.Blank cell shows variant not to be enough to the horizontal expression allowing to analyze further.The activity of variant to amylopectin substrate (Fig. 8) and pullulan substrate (Fig. 9) in position 2,3,88,92,251,252,253,254,256,308,316,317,318,320,321,357,400,402 and 404 (see SEQ ID NO:4) with displacement is tested.

The value of data is with the log of performance index (PI) (2) report, in the case, negative number representation reduces with the amylase phase specific activity at specified location with wt amino acid residue, and positive number represents and to raise with the amylase phase specific activity at specified location with wt amino acid residue, and wherein each unit change represents the coefficient of 2.Strengthen and the data of the sudden change of the activity of substrate are shown with boldface letter.The quantity (No.) of the variant tested, maximum log (2) PI value (Max), log (2) PI is greater than the quantity (#<-2) of the variant of-2, log (2) PI is greater than the per-cent (%>-2) of the variant of 2, log (2) PI is greater than the quantity (#<-1) of the variant of-1, log (2) PI is greater than the per-cent (%>-1) of the variant of-1, log (2) PI is greater than the quantity (#<0) of the variant of 0, log (2) PI is greater than in the per-cent (%>0) of the variant of 0 and the row of average log (2) PI value (Ave.) below and points out.

The activity to amylopectin substrate is improved in the displacement of position 92,251,254,256,317,318,320 and 321 (see SEQ IDNO:4).The activity to amylopectin substrate is improved in the displacement of position 253 and 256.Improving the particular permutation of the activity of amylopectin substrate is S92L, R251A, R251C, R251D, R251E, R251F, R251G, R251H, R251L, R251M, R251N, R251Q, R251S, R251T, and R251W, T254H, T254K, T254W, and T254Y, K256A, K256E, K256T, and K256V, S317C and S317R, N318A N318F, N318H, N318K, N318Q, and N318R, T320A, T320H, T320M, T320N, and T320P, and K321D, K321G, K321I, and K321T.R251 and K256 is replaced as the rising that other amino-acid residues generally cause the activity to amylopectin substrate.

Do not cause the reduction of improvement to the activity of amylopectin substrate or activity in the displacement of position N88, A252, A253, N308, A316, S357, T400, R402 and D403, and result in express in the displacement of position N4, G5, T38, N93, G94, I95, Q96, V97, Y230, G255, V315, P319, A322, L354, T355, R356, G359, Y396, A397, Y398, G399 and Q401 and reduce (see SEQ ID NO:4).

For all objects, all references cited herein is incorporated herein by reference all in full.

Claims (31)

1., for generation of a method for variant alpha amylase polypeptide, comprising:

The amino-acid residue place of aminoacid sequence in starch engagement groove to parent family 13 α-amylase polypeptide introduces sudden change;

Wherein said starch engagement groove is by the 7th ring between alpha-helix and Article 8 beta chain in the 6th ring between alpha-helix and Article 7 beta chain, A structural domain in the amino-acid residue in the alpha-helix in A structural domain before Article 1 beta chain, A structural domain, and the ring connecting A structural domain and C-structure territory is formed; And

Wherein described sudden change changes the combination of starch and described variant alpha amylase polypeptide compared with described parent alpha-amylase polypeptide.

2. method according to claim 1, wherein said starch engagement groove correspond to amino-acid residue 1-6,36,38,91-97,224-226,249-257,278-282,309-320,354-359,391 and 395-402, relevant numbering is see SEQ ID NO:2; Amino-acid residue 1-6,37,39,92-98,227-229,252-260,281-285,312-323,357-362,391 and 395-402, relevant numbering is see SEQ ID NO:3; Or amino-acid residue 1-4,36,38,91-97,226-228,251-259,280-284,311-322,356-361,363,391 and 395-402, relevant numbering is see SEQ ID NO:4.

3., according to method in any one of the preceding claims wherein, wherein said sudden change is in the amino-acid residue corresponding to amino-acid residue 92,251,254,256,317,318,320 or 321, and relevant numbering is see SEQ ID NO:4.

4., according to method in any one of the preceding claims wherein, wherein said sudden change is corresponding to the position of R251 or K256 by the different radical amino acid replacement of wildtype residues, and relevant numbering is see SEQ ID NO:4.

5. the method according to any one of aforementioned claim 1-3, wherein said sudden change is replaced by wildtype residues L in the position corresponding to the position 92 in SEQ ID NO:4, corresponding to the position of the position 251 in SEQ ID NO:4 by wildtype residues A, C, D, E, F, G, H, L, M, N, Q, S, T or W replaces, and is corresponding to the position of the position 254 in SEQ IDNO:4 by wildtype residues H, K, W or Y replaces, and is corresponding to the position of the position 256 in SEQ ID NO:4 by wildtype residues A, E, T or V replaces, and is replaced by wildtype residues C or R in the position corresponding to the position 317 in SEQ ID NO:4, is corresponding to the position of the position 318 in SEQ ID NO:4 by wildtype residues A, F, H, K, Q or R replaces, and is corresponding to the position of the position 320 in SEQ ID NO:4 by wildtype residues A, H, M, N or P replaces, or is corresponding to the position of the position 321 in SEQ ID NO:4 by wildtype residues D, G, I or T replaces.

6. the method according to any one of aforementioned claim 1-3, wherein said sudden change corresponds to S92L, R251A, R251C, R251D, R251E, R251F, R251G, R251H, R251L, R251M, R251N, R251Q, R251S, R251T, or R251W, T254H, T254K, T254W, or T254Y, K256A, K256E, K256T, or K256V, S317C or S317R, N318A N318F, N318H, N318K, N318Q, or N318R, T320A, T320H, T320M, T320N, or T320P, and/or K321D, K321G, K321I, or K321T, relevant numbering is see SEQ ID NO:4.

7. according to method in any one of the preceding claims wherein, wherein said variant is also included in the wt amino acid residue of the one or more positions corresponding to N88, A252, A253, N308, A316, S357, T400, R402 and D403, and relevant numbering is see SEQID NO:4.

8. the method according to any one of aforementioned claim 1-7, wherein said variant is also included in the wt amino acid residue of the one or more positions corresponding to N4, G5, T38, N93, G94, I95, Q96, V97, Y230, G255, V315, P319, A322, L354, T355, R356, G359, Y396, A397, Y398, G399 and the Q401 in SEQ ID NO:4, and relevant numbering is see SEQ ID NO:4.

9. the method according to any one of aforementioned claim 1-8, wherein said variant is also included in N, the A in position 252, the A in position 253, the N in position 308, the A in position 316, the S in position 357, the T in position 400, the R in the position 402 or D in position 403 of position 88, and relevant numbering is see SEQ ID NO:4.

10. the method according to any one of aforementioned claim 1-9, wherein said variant is also included in the N of position 4, at the G of position 5, at the T of position 38, at the N of position 93, at the G of position 94, at the I of position 95, at the Q of position 96, at the V of position 97, at the Y of position 230, at the G of position 255, at the V of position 315, at the P of position 319, at the A of position 322, at the L of position 354, at the T of position 355, at the R of position 356, at the G of position 359, at the Y of position 396, at the A of position 397, at the Y of position 398, at the G of position 399, with the Q in position 401, relevant numbering is see SEQID NO:4.

11. according to method in any one of the preceding claims wherein, wherein uses cyclodextrin to measure relative starch and combines.

12. according to method in any one of the preceding claims wherein, wherein said variant have with SEQID NO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

13. according to method in any one of the preceding claims wherein, wherein said parent have with SEQID NO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

14. according to method in any one of the preceding claims wherein, and wherein said variant shows the starch liquefacation of improvement, mashing or clean-up performance compared with described parent.

15. according to method in any one of the preceding claims wherein, and wherein said variant shows the hydrolytic activity strengthened amylopectin substrate compared with described parent.

The variant alpha amylase polypeptide that 16. 1 kinds of methods by aforementioned claim produce.

The variant of 17. 1 kinds of parent family 13 α-amylase polypeptide, comprises sudden change in starch engagement groove;

Wherein said starch engagement groove is by the 7th ring between alpha-helix and Article 8 beta chain in the 6th ring between alpha-helix and Article 7 beta chain, A structural domain in the amino-acid residue in the alpha-helix in A structural domain before Article 1 beta chain, A structural domain, and the ring connecting A structural domain and C-structure territory is formed;

Wherein described sudden change changes the combination of starch and described variant alpha amylase polypeptide compared with described parent alpha-amylase polypeptide.

18. variant alpha amylase polypeptide according to claim 17, wherein said starch engagement groove correspond to amino-acid residue 1-6,36,38,91-97,224-226,249-257,278-282,309-320,354-359,391 and 395-402, relevant numbering is see SEQ IDNO:2; Amino-acid residue 1-6,37,39,92-98,227-229,252-260,281-285,312-323,357-362,391 and 395-402, relevant numbering is see SEQ IDNO:3; Or amino-acid residue 1-4,36,38,91-97,226-228,251-259,280-284,311-322,356-361,363,391 and 395-402, relevant numbering is see SEQ ID NO:4.

19. variant alpha amylase polypeptide according to aforementioned claim 17 or 18, wherein said sudden change is in the amino-acid residue corresponding to amino-acid residue 92,251,254,256,317,318,320 or 321, and relevant numbering is see SEQ ID NO:4.

20. variant alpha amylase polypeptide according to any one of aforementioned claim 17-19, wherein said sudden change is corresponding to the position of R251 or K256 by the different radical amino acid replacement of wildtype residues, and relevant numbering is see SEQ ID NO:4.

21. variant alpha amylase polypeptide according to any one of aforementioned claim 17-19, wherein said sudden change is replaced by wildtype residues L in the position corresponding to the position 92 in SEQ ID NO:4, corresponding to the position of the position 251 in SEQ ID NO:4 by wildtype residues A, C, D, E, F, G, H, L, M, N, Q, S, T or W replaces, and is corresponding to the position of the position 254 in SEQ ID NO:4 by wildtype residues H, K, W or Y replaces, and is corresponding to the position of the position 256 in SEQ ID NO:4 by wildtype residues A, E, T or V replaces, and is replaced by wildtype residues C or R in the position corresponding to the position 317 in SEQ ID NO:4, is corresponding to the position of the position 318 in SEQ ID NO:4 by wildtype residues A, F, H, K, Q or R replaces, and is corresponding to the position of the position 320 in SEQ ID NO:4 by wildtype residues A, H, M, N or P replaces, or is corresponding to the position of the position 321 in SEQ ID NO:4 by wildtype residues D, G, I or T replaces.

22. variant alpha amylase polypeptide according to any one of aforementioned claim 17-19, wherein said sudden change corresponds to S92L, R251A, R251C, R251D, R251E, R251F, R251G, R251H, R251L, R251M, R251N, R251Q, R251S, R251T, or R251W, T254H, T254K, T254W, or T254Y, K256A, K256E, K256T, or K256V, S317C or S317R, N318AN318F, N318H, N318K, N318Q, or N318R, T320A, T320H, T320M, T320N, or T320P, or K321D, K321G, K321I, or K321T, relevant numbering is see SEQ ID NO:4.

23. variant alpha amylase polypeptide according to any one of aforementioned claim 17-22, wherein said variant is also included in the wt amino acid residue of the one or more positions corresponding to N88, A252, A253, N308, A316, S357, T400, R402 and/or D403, and relevant numbering is see SEQ ID NO:4.

24. variant alpha amylase polypeptide according to any one of aforementioned claim 17-23, wherein said variant is also included in the wt amino acid residue of the one or more positions corresponding to N4, G5, T38, N93, G94, I95, Q96, V97, Y230, G255, V315, P319, A322, L354, T355, R356, G359, Y396, A397, Y398, G399 and/or the Q401 in SEQ ID NO:4, and relevant numbering is see SEQ ID NO:4.

25. variant alpha amylase polypeptide according to any one of aforementioned claim 17-24, wherein said variant is also included in N, the A in position 252, the A in position 253, the N in position 308, the A in position 316, the S in position 357, the T in position 400, the R in the position 402 and/or D in position 403 of position 88, and relevant numbering is see SEQ ID NO:4.

26. variant alpha amylase polypeptide according to any one of aforementioned claim 17-25, wherein said variant is also included in the N of position 4, at the G of position 5, at the T of position 38, at the N of position 93, at the G of position 94, at the I of position 95, at the Q of position 96, at the V of position 97, at the Y of position 230, at the G of position 255, at the V of position 315, at the P of position 319, at the A of position 322, at the L of position 354, at the T of position 355, at the R of position 356, at the G of position 359, at the Y of position 396, at the A of position 397, at the Y of position 398, at the G of position 399, and/or the Q in position 401, relevant numbering is see SEQ ID NO:4.

27. variants according to any one of aforementioned claim 17-26, wherein said variant have with SEQ ID NO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

28. variants according to any one of aforementioned claim 17-27, wherein said parent have with SEQ ID NO:1,2,3,4 or 5 at least 60%, at least 70%, the amino acid sequence identity of at least 80% or at least 90%.

29. variants according to any one of aforementioned claim 17-28, wherein said variant shows the starch liquefacation of improvement, mashing or clean-up performance compared with parent.

30. variants according to any one of aforementioned claim 17-29, wherein said variant shows the hydrolytic activity strengthened amylopectin substrate compared with described parent.

31. 1 kinds of compositions, comprise the variant alpha amylase polypeptide according to any one of aforementioned claim 17-30 and at least one preparaton.